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The expression level of CB1 and CB2 receptors determines their efficacy at inducing apoptosis in astrocytomas.

Cudaback E, Marrs W, Moeller T, Stella N - PLoS ONE (2010)

Bottom Line: Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT.Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.

Principal findings: We generated astrocytoma subclones that express set levels of CB(1) and CB(2), and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

Significance: The high expression level of CB(1) and CB(2) receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB(1) and CB(2) receptors, yet still activate ERK1/2.

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Activation of CB1 and CB2 receptors increases ERK1/2 phosphorylation in all DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and ERK1/2 phosphorylation quantified by Western blot analysis. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in ERK1/2 phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6–8 independent experiments expressed as % of vehicle (i.e. level of ERK1/2 phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Note that basal ERK1/2 phosphorylation did not vary significantly over time (Figure S1a). In (a), (*) = p<0.05 and (**) = p<0.01 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test.
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pone-0008702-g001: Activation of CB1 and CB2 receptors increases ERK1/2 phosphorylation in all DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and ERK1/2 phosphorylation quantified by Western blot analysis. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in ERK1/2 phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6–8 independent experiments expressed as % of vehicle (i.e. level of ERK1/2 phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Note that basal ERK1/2 phosphorylation did not vary significantly over time (Figure S1a). In (a), (*) = p<0.05 and (**) = p<0.01 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test.

Mentions: Because previous studies performed on astrocytoma cell lines have implicated ERK1/2 signaling in the therapeutic actions of cannabinoids [23], we sought to determine whether different expression levels of CB receptors would affect their coupling to this signal transduction pathway. To do so, we treated each subclone with CP-55,940 (1 µM, maximally efficacious concentration [17], [24]) and measured ERK1/2 phosphorylation by Western blot analysis. We found that CP-55,940 induced a significant increase in ERK1/2 phosphorylation above vehicle-treated controls in all six subclones (Figure 1a). All these responses were receptor-mediated since they were significantly attenuated by either the CB1 antagonist SR141716A, or the CB2 antagonist SR144528, respectively (Figure 1a). Furthermore, CP-55,940 did not affect ERK1/2 phosphorylation in wild-type DBT cells (Figure S1a). The magnitude and kinetics of CP-55,940-induced ERK1/2 phosphorylation mediated in each subclone were remarkably similar despite a 10-fold difference in receptor expression level between the CB1-low and the CB1-high subclones, and between the CB2-low and the CB2-high subclones (Figure 1b,c). The only differences that we noted between CB1- and CB2-mediated regulation of ERK1/2 phosphorylation were that CP-55,940 induced a slightly slower and sustained increase in ERK1/2 phosphorylation in CB1-expressing subclones (peak at 5 min returning to basal by 20–30 min) compared to CB2-expressing subclones (peak at 3 min returning to basal by 10–20 min) (Figure 1b,c). These results show that CB1 and CB2 receptors heterologously expressed in DBT cells are functional for they regulate ERK1/2 phosphorylation. They also show that the efficacy of this regulation is largely independent of receptor expression levels and receptor subtype.


The expression level of CB1 and CB2 receptors determines their efficacy at inducing apoptosis in astrocytomas.

Cudaback E, Marrs W, Moeller T, Stella N - PLoS ONE (2010)

Activation of CB1 and CB2 receptors increases ERK1/2 phosphorylation in all DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and ERK1/2 phosphorylation quantified by Western blot analysis. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in ERK1/2 phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6–8 independent experiments expressed as % of vehicle (i.e. level of ERK1/2 phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Note that basal ERK1/2 phosphorylation did not vary significantly over time (Figure S1a). In (a), (*) = p<0.05 and (**) = p<0.01 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test.
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pone-0008702-g001: Activation of CB1 and CB2 receptors increases ERK1/2 phosphorylation in all DBT subclones.DBT subclones stably expressing cannabinoid receptors were expanded in 24-well plates, incubated with CP-55,940 (CP, 1 µM), and ERK1/2 phosphorylation quantified by Western blot analysis. (a) CB1- and CB2-expressing subclones were incubated with CP for 5 and 3 min, respectively (white bars). When testing the effect of antagonists, cells were pretreated with SR141716A (5 µM) and SR144528 (3 µM) added 10 min before CP (black bars). (b,c) Kinetics of CP-induced increase in ERK1/2 phosphorylation in CB1-low, CB1-high, CB2-low and CB2-high subclones. Data = mean±s.e.m. of 6–8 independent experiments expressed as % of vehicle (i.e. level of ERK1/2 phosphorylation when treated with vehicle, i.e. 0.1% DMSO. Note that basal ERK1/2 phosphorylation did not vary significantly over time (Figure S1a). In (a), (*) = p<0.05 and (**) = p<0.01 significantly different from the response in the presence of inhibitor, ANOVA followed by Bonferroni's post-test. In (b,c), (*) and (#) = p<0.05, and (**) and (##) = p<0.01 significantly different from vehicle at corresponding time point, ANOVA followed by Bonferroni's post-test.
Mentions: Because previous studies performed on astrocytoma cell lines have implicated ERK1/2 signaling in the therapeutic actions of cannabinoids [23], we sought to determine whether different expression levels of CB receptors would affect their coupling to this signal transduction pathway. To do so, we treated each subclone with CP-55,940 (1 µM, maximally efficacious concentration [17], [24]) and measured ERK1/2 phosphorylation by Western blot analysis. We found that CP-55,940 induced a significant increase in ERK1/2 phosphorylation above vehicle-treated controls in all six subclones (Figure 1a). All these responses were receptor-mediated since they were significantly attenuated by either the CB1 antagonist SR141716A, or the CB2 antagonist SR144528, respectively (Figure 1a). Furthermore, CP-55,940 did not affect ERK1/2 phosphorylation in wild-type DBT cells (Figure S1a). The magnitude and kinetics of CP-55,940-induced ERK1/2 phosphorylation mediated in each subclone were remarkably similar despite a 10-fold difference in receptor expression level between the CB1-low and the CB1-high subclones, and between the CB2-low and the CB2-high subclones (Figure 1b,c). The only differences that we noted between CB1- and CB2-mediated regulation of ERK1/2 phosphorylation were that CP-55,940 induced a slightly slower and sustained increase in ERK1/2 phosphorylation in CB1-expressing subclones (peak at 5 min returning to basal by 20–30 min) compared to CB2-expressing subclones (peak at 3 min returning to basal by 10–20 min) (Figure 1b,c). These results show that CB1 and CB2 receptors heterologously expressed in DBT cells are functional for they regulate ERK1/2 phosphorylation. They also show that the efficacy of this regulation is largely independent of receptor expression levels and receptor subtype.

Bottom Line: Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT.Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB(1) and CB(2) receptors mediate this therapeutic effect is unclear.

Principal findings: We generated astrocytoma subclones that express set levels of CB(1) and CB(2), and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB(1), CB(2) and AKT, but still through a mechanism involving ERK1/2.

Significance: The high expression level of CB(1) and CB(2) receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB(1) and CB(2) receptors, yet still activate ERK1/2.

Show MeSH
Related in: MedlinePlus